The present invention relates generally to a plant propagation system, and more particularly to an aseptic propagation system and process for promoting the growth of plant tissue into transplants.
Micropropagation, sometimes referred to as tissue culture propagation, is the process of growing new plants from a piece of plant tissue that has been extracted from a parent plant with desired characteristics. Micropropagation has recently grown in popularity as a preferred plant propagation technique for a wide range of horticultural crops because of high production efficiency and greater uniformity of the resulting plants. The process results in the mass reproduction of plants having certain desirable characteristics since substantially all of the plants produced are genetically identical to and have all of the desirable traits of the parent. Micropropagation is an especially useful process for genetically engineered plants, high-value transplants, seedless fruits and vegetables, certified disease free plant material and all other plants that cannot be produced from seed economically or uniformly.
In general terms, micropropagation typically includes first selecting a parent plant. The parent plant should be healthy and should have the desired traits that are needed in the next generation plants. A tissue sample is then extracted from the parent. The sample is typically meristematic tissue which is undifferentiated tissue capable of dividing and giving rise to other meristemic tissue as well as specialized tissue types. Meristematic tissue is found in growth areas such as at the tips of stems or at lateral buds. The tissue sample (explant) is disinfested and then placed in a controlled environment and supplied essential nutrients for promoting growth.
Growth of the plant tissue sample into a small plant occurs in four commonly referred to stages. First, growth of the explant is established in a sterile environment. Second, high prolifferation of explant is promoted by repeated selection of small pieces of tissue containing vegetative buds, or other specialized propagative structures (e.g. bulbets, protocorm-like bodies (PLB), microtubers, somatic embryos). The third stage involves forming a shoot from the vegetative bud. The fourth stage involves forming a root on the shoot, thereby completing the development of a whole plant from the plant tissue.
During the first and second stage of growth, the plant tissue is made up of small rapidly dividing cells with high metabolic requirements for energy. The tissue is incapable of carrying out adequate photosynthesis to meet this high demand.
Consequently, initial growth of the tissue is done heterotrophically. Heterotrophic growth is where the organism obtains nourishment and energy from the ingestion and breakdown of organic matter. During this phase, the plant tissue is typically not exposed to light and is fed a growth medium containing organic carbon. The organic carbon is usually obtained from sugars such as sucrose.
In the third stage of growth, leaves and shoots expand and the plant tissue becomes more capable of photosynthesizing. The plant tissue, when exposed to light, gases, water and essential nutrients, derives energy photoautotrophically through the process of photosynthesis. Photoautotrophic growth is where an organism synthesizes organic nutrients by deriving energy from light. In other words, during autotrophic growth, the plant tissue is capable of making its own food which it cannot do adequately during the other stages.
Traditionally, tissue culture propagation has been done on agar or semisolid mediums for providing nutrients and organic carbon to the plant material. The nutrient mediums have been contained in small glass or plastic containers open to the atmosphere for allowing needed gas exchange. However, many problems have been encountered using these techniques. For instance, the largest of these problems has been contamination. Microorganisms such as bacteria, fungus, viruses, molds, yeast or other small plants, which thrive on the organic compounds in the mediums, can attack and kill or inhibit the growth of vulnerable plant tissue samples. In order to prevent contamination, the tissue samples have to be placed in a sterile and controlled environment. As such, all work has typically been confined to the laboratory.
Another disadvantage to using agar or semisolid mediums is the expense or production costs involved in growing the plants. First, high costs are involved in maintaining stringent aseptic environments as described above. The facilities and equipment needed are also expensive necessities. Further, using agar or semisolid liquid mediums requires large amounts of manual labor. For instance, the growing plantlets must be frequently transferred to new vessels with fresh media. This work is very labor intensive because the fragile plants are typically embedded in the spent media and need to be carefully removed. These multiple transfers also limit the ability to automate the system.
Recently, many attempts have been made to develop a plant tissue propagation system that does not require growing plants on agar or semisolid mediums. Instead, the plants are fed a liquid nutrient solution. For instance, U.S. Pat. No. 5,225,342 to Farrell discloses an artificial replacement for the vascular and support functions normally provided by the root system of a plant. The apparatus includes placing a totipotent plant cell on an artificial xylem surface. Xylem is the principal water and mineral conducting tissue in vascular plants. Nutrient solutions are applied in a manner which encourages the growth of the cell to form aerial portions of the plant and which essentially prohibits the growth of plant roots.
In one embodiment, a growth chamber for a vascular plant having xylem and phloem tissue is provided. The chamber is constructed from a high impact polystyrene or polycarbonate material and has three trays for holding various liquids. Inside the chamber, a plurality of totipotent plant cells or explant tissue is inserted through incisions in a sponge which is placed on an artificial xylem surface. A nutrient solution, circulated through the lower tray, is fed to the plant tissue through the artificial xylem. The artificial xylem is preferably constructed of small tubules made from polypropylene. A rinse solution is introduced into the upper tray where it falls onto the sponge where the plant tissue has been inserted. The rinse solution then flows through the sponge to the middle tray where it exits the chamber. Preferably, the rinse media is a sterile, deionized aqueous solution adjusted to the physiological pH of the plant, but any solution which is isotonic and capable of removing endotoxins or waste material exuded by the plant tissue into the sponge is acceptable. Further, the chamber includes a top cover which has a carbon dioxide inlet and a microbial shield-water vapor diffusion membrane. The membrane, preferably made from polyethylene, permits gas transfer without the introduction of microbial contamination into the chamber.
U.S. Pat. Nos. 5,171,683 and 4,908,315, both to Kertz, disclose an integument and method for microbe propagation and tissue culturing. Here, a sealed integument is made of a semipermeable and translucent membrane which allows light transmission and gas exchange but seals out biological contaminants. A plant, seeds, or plant tissue is placed inside the sealed integument along with a growth medium such as soil or a liquid media. The integument is liquid impermeable so that a liquid or semisolid growth medium cannot escape and dry out the plant. The integument is made from a polyethylene material.
A method and apparatus for culturing autotrophic plants from heterotrophic plant material is disclosed in Timmis et al., U.S. Pat. No. 5,119,588. In this patent, plant material is embedded in a plug of particulate medium having soil-like properties. The plug should be sterile and may contain water, mineral nutrients or plant hormones. The plug and plant material are then placed in a bag which is preferably made of a material which allows the passage of light and gases necessary for plant growth and development from the ambient environment to the interior of the bag. The bag preferably also passes water vapor at a slow rate from the interior of the bag to the ambient environment for humidity control inside the bag. Candidate bag materials include high density polyethylene, polypropylene, and fluorinated ethylenepropylene.
Other plant growing systems are discussed and disclosed in U.S. Pat. No. 5,212,906 to Okuno et al., U.S. Pat. No. 5,184,420 to Papadopoulis et al., U.S. Pat. No. 5,088,231 to Kertz, and U.S. Pat. No. 5,049,505 to Sei.
Although disclosing an assortment of plant propagation and growing systems, the prior art still has its drawbacks and deficiencies. For instance, some of the prior art fails to provide a method or apparatus that is truly effective at preventing contamination of the growing plant material. For instance, in many prior art devices the plant material is vulnerable to attack once the growth medium becomes infected with harmful microorganisms. Also, many of the prior art methods fail to provide an efficient way to replace or replenish the plant growth medium once it becomes spent. Further, once sealed in a container, problems have been encountered in the past in getting the small plantlets to photosynthesize.